INTERNATIONAL RESEARCH JOURNAL OF PHARMACY
www.irjponline.com
ISSN 2230 – 8407
Research Article
EXPLORATION OF BIO-ACTIVE PHYTOCOMPONENTS AND EVALUATION OF
ANTI-MICROBIAL POTENTIAL OF
STRYCHNOS POTATORUM
Pitchiah Kumar Murugan
1*, Kaliyamurthi Venkatachalam
21
Part time Research Scholar, Centre for Research and Development, Prist University, Thanjavur, Tamil Nadu, India
2Assistant Professor, Department of Basic sciences, Fisheries College and Research Institute, Thoothukudi,
Tamil Nadu, India
*Corresponding Author Email: [email protected]
Article Received on: 12/06/19 Approved for publication: 12/07/19
DOI: 10.7897/2230-8407.1009283
ABSTRACT
Research interest towards herbal medicine grabs major attention in recent times due to its innumerable role in the process of drug discovery. It is evident that traditional medicines contribute as a therapeutic ailment for treating dreadful disease. Present investigation aimed at exploring the major phytocomponents present in the herb Strychnos potatorum (SP) and to screen its possible anti-microbial activity against selective pathogens. Ethanol (EESP), Aqueous (AESP) and Hydro alcoholic (HAESP) extracts were screened using biochemical, GCMS and FTIR analysis. Outcome of the study clearly signifies the presence of bioactive components such as alkaloids, flavonoids, tannins, phenols and saponin in the extracts. Results of GCMS justify the existence of ascorbic, oleic and erucic acid in EESP. Further higher level of active components was reports in EESP. FTIR study evidents the presence of major active functional groups in the extracts. Anti-microbial evaluation reveals that the EESP exhibits maximum zone of inhibition against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Enterococcus faecalis and Klebsiella pneumonia. Present study provides an evidence-based data’s with respect to the category and nature of phytocomponents present in the herb Strychnos potatorum and hence the promising anti-microbial activity may due to its biologically active phyto therapeutics which may render beneficial activity against infectious and cardiovascular diseases subjected to proper preclinical justification in near future.
Keywords: Herbal medicines, Strychnos potatorum, Phyto components, Anti-microbial, Oleic acid, GCMS, FTIR.
INTRODUCTION
Drugs derived from natural sources have significant contribution in the prevention and treatment of disease to mankind. In many developing countries, traditional and folklore medicines are the
first drug of choice for their primary healthcare systems.1,2 Herbs
are widely used and well explored in most of the traditional
medicine and their curative potentials are well documented3.
World Health Organization estimates that nearly 80% of the people in developing countries rely on traditional remedies such as medicinal herbs and folklore medicines for their health care benefits. The use of medicinal plants has been developed over a long period of time and now plays a critical role in favorable health outcomes. Today, a number of pharmaceutical products currently approved by the Food and Drug Administration (FDA) have originated from plants; natural products (and their derivatives and analogs) represent over 50% of all drugs in clinical use.4-7
The number of multi-drug resistant microbial strains and the appearance of such strains had significantly reduced susceptibility to antibiotics are continuously increasing. This scenario has been attributed to indiscriminate use of
broad-spectrum antibiotics, immunosuppressive agent.8 Pharmaceutical
industries are at dire need of phyto therapeutics with broad spectral anti-microbial property and offers minimal resistance when compare to conventional antibiotics.
Cardiovascular diseases (CVDs) become most prevalent cause of human morbidity and mortality globally. According to the research survey by global burden of disease Study it was estimated that nearly 29.6% of all deaths worldwide were caused by CVDs in 2010. It is estimated that the number of people that die from CVDs, mainly from heart disease and stroke, will
increase to more than 24 million by 2030.9-12
Strychnos potatorum (SP) is a medium-sized, glabrous tree. Stem is fluted and covered with black, thick, square to rectangular
scales13. Seeds of Strychnos potatorum possess diuretic, anti
diarrheal and anti diabetic activity. Mannogalactans isolated from
the seeds of Strychnos potatorum reported to have
anti-hypercholesterolemic activity in experimental rats.14 Paste of
seed has been consumed internally along with tender coconut
milk in the management of urinary retention and other disorder15.
As per the literature research it was strongly evident that the plant
Strychnos potatorum has wide range of pharmacological activity
which includes Anti-diabetic16, Anti-inflammatory17,18,
Anti-ulcerogenic19, Hepatoprotective20, Antioxidant activity,
Anti-arthritic21, Anti-nociceptive22, Anti-pyretic, Anti-diarrheal23,
Diuretics24 and Antimicrobial properties25.
Oxidative stress becomes major contributing factor for majority of the chronic diseases such as atherosclerosis, hematological and neurodegenerative disorders. Generation of free radicals due to oxidative stress factors associated with inflammation and other
diseases became major health issues in recent years.26,27
diseases. It has been already reported in our previous work that extract of EP possess significantly higher level of anti-oxidant
property in the tested medium.28 Hence in the present
investigation aimed at evaluating the phytochemical and
anti-microbial potential of the medicinal herb Strychnos
potatorum using high throughput analytical techniques like GCMS and FTIR.
MATERIALS AND METHODS
Plant collection
Fruits of Strychnos potatorum were collected and were identified
by expert taxonomist. Plant materials were then washed separately with fresh water to remove dirt and other contaminants. Epicarp of the fruits was separated and was shade-dried for several days with occasional sun drying. The shade-dried materials were ground into coarse powder by a grinding machine and the materials were stored at room temperature for future use.
Preparation of the extract
About 1 kg of coarse powdered epicarp of Strychnos potatorum
was passed through a 60 No mesh sieve. Air dried powdered drug was extracted with the following solvents like Water, Ethanol and mixture of Ethanol: water (6:4) (hydro-alcoholicextract) by using soxhlet extraction. Then the extracts obtained such as aqueous
extract of Strychnos potatorum (AESP), Ethanol extract of
Strychnos potatorum (EESP) and Hydro-alcoholic extract of
Strychnos potatorum (HAESP) was filtered, concentrated by rotary vacuum pump to get the solid mass.
Preliminary Qualitative Phytochemical Analysis
Qualitative biochemical analysis was carried for identification of category of phytocomponents in all three extracts. Presence of steroids identified using fluorescence in the presence of sulphuric acid. Alkaloids by using mayer's test. Identification of flavonoids was carried out by ammonia test. Glycosides and triterpenoids using borntrager’s test and liebermann–burchard test. Ferric chloride and lead acetate test were used for identification of tannins and phenols, further presence of proteins and saponins
identified using biuret and foam test.29
Quantitative Estimation
Estimation of Total Flavonoid
Total flavonoid content in the extracts was determined using aluminum chloride method. In this method Quercetin was used as standard and flavonoid contents were measured as quercetin equivalent. For this purpose, the calibration curve of quercetin was drawn. 1 ml of standard or extract solution was taken into 10 ml volumetric flask, containing 4 ml of distilled water. 0.3 ml of
5% NaNO2 added to the flask. After 5 min, 0.3 ml 10% AlCl3 was
added to the mixture. At the 6th min add 2 ml of 1M NaOH was
added and volume made up to 10 ml with distilled water. The absorbance was noted at 510 nm using UV-Visible
spectrophotometer.30
Estimation of Alkaloid
5 g of the test sample was weighed into a 250 ml beaker and 200 ml of 10% acetic acid in ethanol was added and covered and allowed to stand for 4 hr. This was filtered and the extract was concentrated on a water bath to one-quarter of the original volume. Concentrated ammonium hydroxide was added drop wise to the extract until the precipitation was complete. The
whole solution was allowed to settle and the precipitated was collected and washed with dilute ammonium hydroxide and then filtered. The residue is the alkaloid, which was dried and
weighed.31
Estimation of Tannin
The tannin content of the extracts was determined using Folin– Ciocalteu assay. Aliquot extract of the test dru of 100 µL was added to 750 µL of distilled water, 500 µL Folin–Ciocalteu
reagents and 1000 µL of 35 % sodium carbonate (Na2CO3). The
mixture was shaken vigorously after diluting to 10 mL of distilled water. The mixture was incubated for 30 min at room temperature and read at 725 nm. Distilled water was used as blank. Tannic acid standard solutions were prepared, and standard calibration curve was plotted with varying concentration. The total tannins content was expressed as Tannic acid mg/gm, as calculated from
the prepared standard curve.32
Determination of total Phenol content
The total phenol content of the extracts was determined using Folin–Ciocalteu reagents with analytical grade Gallic acid as the standard. 1 ml of sample was added to deionized water (10 ml) and Folin–Ciocalteu phenol reagents (1 ml). After 5 minutes, 20% sodium carbonate (2 ml) was added to the mixture. After being kept in total darkness for 1 hr, the absorbance was measured at 750 nm using a spectrophotometer. Amounts of total Phenol was calculated using Gallic acid calibration curve. The results were expressed as Gallic acid equivalents (GAE) mg/g of dry plant matter.33
Fourier Transform – Infra Red Spectroscopy Study
Fourier Transform – Infra Red Spectroscopy Study (FTIR) IR data acquired with Shimadzu –IR affinity 1S portal, Software: Agilent Resolutions pro. About 20 mg of the test sample was taken on a micro spatula and grounded well with required quantity of KBr salt. Sample admixed with KBr with trituration aided by mortar and pestle until to get a uniform fine powder of sample- KBr mixture. Further mixture was loaded in pellet die and subjected to 5000-10,000 psi in pelletizer. Resulting pellet was placed in FTIR sample holder and expose to IR radiation to get the spectra.34
GC-MS Analysis
GC-MS analysis of the extracts were carried out using Agilent 7890 B GC connected to 5977A MSD, NIST Ver.2.1 MS data library. GC-MS becomes a reliable tool for identifying the presence of volatile components including phytosterols in the
herbal extracts and fractions.35
Anti- Microbial Activity
Disc diffusion method adopted for antimicrobial profiling of the sample EESP at the concentration varying from 100 to 300 µg. MHB broth used for culturing specific target pathogens such as
Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Enterococcus faecalis and Klebsiella pneumonia and standardized for the period of 24 h. Sub cultures of each strains inoculated in plate containing MHA medium. Test drug along with respective standards Streptomycin (20 µg) for anti-bacterial and Fluconazole (20 g) for anti-fungal injected on sterile disc positioned on each quadrant of the petri plates. Incubation allowed for the period of 24 h (bacterial) and 72 h (fungal). Inhibitory zone around the disc was measured and expressed in
Table 1: Qualitative Phytochemical analysis of AESP, EESP and HAESP
Test Sample Steroids Alkaloids Flavonoids Glycosides Terpenoid Tannins Poly phenol Protein Saponin
EESP - + + - + + + - +
AESP - + + - - + + + +
HAESP - + + - - + + + +
Table 2: Qualitative Phytochemical analysis of AESP, EESP and HAESP
Phytoconstituents AESP EESP HAESP Total flavonoids (quercetin mg/gm) 0.77 ± 0.68 1.60 ± 0.24 1.09 ± 1.3
Total alkaloids (mg/gm) 0.69 ± 0.15 3.11 ± 0.26 1.13 ± 0.07
Total tannins (mg/gm) (Tannic acid mg/gm) 0.23 ± 0.08 0.52 ± 0.03 0.37 ± 0.02
Total Phenol (Gallic acid equivalents (GAE) mg/g) 0.016 ± 0.003 0.044 ± 0.06 0.028 ± 0.02
Mean with 3 replicates ± SD
Figure 1: FT-IR Spectrum of ECP
Table 3: FT-IR Peak Table analysis of ECP
Absorption Peak No Range % Transmittance
1. 3407.64 cm-1 94.88
2. 2924.95 cm-1 98.19
3. 2360.23 cm-1 100.1
4. 1733.87 cm-1 98.16
5. 1635.46 cm-1 96.29
6. 1383.33 cm-1 98.61
7. 1238.40 cm-1 99.42
8. 1426.06 cm-1 98.86
9. 1319.72 cm-1 99.42
10. 1103.09 cm-1 94.59
11. 1055.69 cm-1 94.69
12. 896.99 cm-1 99.40
13. 755.15 cm-1 98.17
14. 656.99 cm-1 96.85
15. 608.18 cm-1 96.67
16. 447.85 cm-1 97.78
Table 4: GCMS Peak Table analysis of AESP
Peak # R. Time Area % Compound Name
1 43.64 23.30 Silane
2 45.62 76.70 Tetrapentacontane
100.00
Figure 3: GCMS Chromatogram of HAESP
Table 5: GCMS Peak Table analysis of HAESP
Peak # R. Time Area % Compound Name
1 6.26 2.08 Decane, 2,3,5,8- tetramethyl-
2 7.06 3.74 1-Iodo-2-methyundecane
3 7.63 20.05 Phenol,3,5- bis (1,1-dimethyethyl)-
4 8.21 13.79 2,3- Di hydrooxazole
5 8.71 3.11 Eicosane
6 8.94 29.30 Decanoic acid
7 9.19 16.43 Capric Acid
8 10.06 8.04 Heptacosane
9 10.86 3.45 Octacosane
100.00
Figure 4: GCMS Chromatogram of EESP
Table 6: GCMS Peak Table analysis of EESP
Peak # R. Time Area % Compound Name
1 9.23 59.75 3-Methylmannoside
2 9.82 7.30 Ascorbic acid
3 10.54 30.42 Oleic acid
4 11.29 2.53 Erucic acid
100.00
Table 7: Zone of Inhibition data on Anti-bacterial activity of EESP against selected Bacterial strains
Sample code Zone of inhibition (mm) Bacillus subtilis Staphylococcus
aureus
Enterococcus faecalis
Pseudomonas aeruginosa
Escherichia coli Klebsiella pneumoniae
100 µg
200 µg
300 µg
100 µg
200 µg
300 µg
100 µg
200 µg
300 µg
100 µg
200 µg
300 µg
100 µg
200 µg
300 µg
100 µg
200 µg
300 µg
EESP 8 10 16 10 12 18 - 7 13 - - 8 8 11 16 - - 7
Streptomycin (20 µg)
25 26 18 12 23 19
Table 8: Zone of Inhibition data on Anti-Fungal activity of EESP against selected fungal strain
Sample code Candida albicans EESP 100 µg 200 µg 300 µg
11 12 16
Fluconazole (20 µg) 20
RESULTS
Qualitative Phytochemical analysis of AESP, EESP and HAESP
The results of the preliminary qualitative phytochemical analysis of AESP, HAESP and EESP justify the presence of alkaloids, flavonoids, tannins, phenols and saponin. In this presence of terpenoids was only evident in the EESP which may be unique for EESP for its expected biological activity. The results were tabulated in Table 1.
Quantitative Phytochemical analysis of AESP, EESP and HAESP
The quantitative estimation of total flavonoid, total alkaloids, total tannins and total phenols in AESP were found to be 0.77, 0.69, 0.23 and 0.016 mg/gm respectively, For EESP, the values were found to be 1.60, 3.11, 0.52 and 0.044 mg/gm. The quantitative estimation of HAESP for of total flavonoid, total alkaloids, total tannins and total phenols were found to be 1.09, 1.13, 0.37 and 0.028 mg/gm respectively. The results were tabulated in Table 2.
FT-IR Analysis of EESP
Strong intense peak at 3407 cm-1 may be due to broad O-H
stretching vibration. Medium peaks at 2924 cm-1 may be due to
presence of N-H str. less intense peak at 2360 cm-1 due to the
presence of O-H and N-H stretching vibration. Peak at 1733 cm-1
due to the presence of Amide (C=O stretching vibration) and peak
at 1635 cm-1 due to C=C stretching vibration; peak at 1383 cm-1
due to N-O stretching vibration and peak at 1426 cm-1 due to
C-H deformation vibration. Peak at 1103 cm-1 due to the presence
of C-O stretching vibration and at 896 cm-1 may be due to N-H
out-of-plane bending vibrations. Sharp peak at 656 cm-1 due to
C=O (conjugated) deformation vibration and at 447 cm-1 may be
due to C-S stretching vibration. The results were tabulated in Table 3 and illustrated in Figure 1.
GCMS analysis of AESP, EESP and HAESP
GCMS analysis of the sample AESP reveals the presence of two independent peaks, whereas for HAESP it revealed the presence of nine prominent peaks and for EESP it shown 4 prominent peaks. The peak 2, 3 and 4 of EESP corresponds to the presence of ascorbic, oleic and erucic acid. The results were tabulated in Table 4 to 6 and illustrated in Figure 2 to 4.
Anti-Microbial Profiling of EESP
EESP has revealed maximum zone of inhibition against
Escherichia coli, Enterococcus faecalis and Klebsiella pneumonia with the inhibitory zone of 16, 13 and 7 mm at similar concentration. As shown in Table 7. Anti-fungal activity of the extract EESP has revealed concentration dependent activity
against Candida albicans with the maximum zone of 16 mm at
the concentration of 300 µg when compared to that of the standard fluconazole. As shown in Table 8.
DISCUSSION
Medicinal herbs are capable of synthesizing secondary
metabolites39 which can be used for the new drugs discovery and
development. Medicinal plant extracts are potential source for the development of new agents effective against many infections disease that are currently difficult to treat. The results of the preliminary phytochemical analysis of the sample AESP, HAESP and EESP reveal the presence of bioactive phyto components such as alkaloids, flavonoids, tannins, phenols and saponin. In this presence of terpenoids was only evident in the EESP which may be unique for EESP for its expected biological activity.
The biomedical potential of the herbs possesses some biologically active components that produce a definite physiological action on the human health. The most important bioactive substances are
alkaloid, tannin, flavonoid and phenolic compounds.40 In the
present study out of three extracts subjected for analysis the EESP has shown increased level of secondary metabolites when compared to aqueous and hydro alcoholic extracts. The quantitative estimation of total flavonoid, total alkaloids, total tannins and total phenols in EESP were found to be 1.60, 3.11, 0.52 and 0.044 mg/gm.
Terpenoids are considerably high potential phyto components which plays very vital role and being the major component of essential oils that make them valuable in the perfumery industry. Within the food industry, the flavoring and preservative potential of essential oils have been well understood along with their
general antimicrobial and antioxidant effects.41-43 Saponins are
other group of secondary metabolites found in most of the medicinal herbs. It was evident though several studies that plant saponin are possess potential anti-obese activities. Saponin could increase fatty acid oxidation and inhibit hepatic glucose
production thereby lowering plasma FFA levels.44 Plant poly
phenols are potential scavengers of free radicals in general. It was well proven fact that regular dietary supplementation of poly phenols aid in prevention of pathological diseases and for the
improvement of human health conditions.45 In the present study
FT-IR analysis of the sample ESP reveals the presence of most significant functional groups such as N-H, O-H, C=O etc.
About 61% of new drugs developed between 1981 and 2002 were based on natural products and they have been very successful,
especially in the areas of infectious disease and cancer.46 Recent
trends, however, show that the discovery rate of active novel
chemical entities is declining.47 Natural products of higher plants
may give a new source of antimicrobial agents with possibly novel mechanisms of action. The effects of plant extracts on bacteria have been studied by a very large number of researchers in different parts of the world. Much work has been done on ethno
medicinal plants in India.48-50 It was observed from the GCMS
analysis of the present investigation that the bioactive component like ascorbic and oleic acid was present exclusively on the EESP. Further it was evident through research that oleic acid, improve
lipid profile and51, maintain a balance of body weight52 and
prevent palmitate-induced mitochondrial dysfunction, insulin
resistance and inflammatory signaling in neuronal cells53 and
CONCLUSION
Strychnos potatorum is a potential medicinal herb which exhibit versatile pharmacological action due to diverse phyto therapeutics present in it. From the results of the present investigation it was concluded that the EESP has significantly higher proportion of phyto components when compare to aqueous and hydro alcoholic extracts. GCMS reveals the presence of ascorbic and oleic acid in EESP. Anti-microbial property of EESP may be due to presence of biologically active phyto components such as alkaloids, flavonoids, tannins, phenols and saponin. Further presence of oleic acid justifies the folklore claim of this herb against cardiovascular and inflammatory disorders. Preclinical investigation will be carried out in future to study its mechanism in suitable animal model.
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Cite this article as:
Pitchiah Kumar and Kaliyamurthi. Exploration of Bio-active
Phytocomponents and Evaluation of anti-microbial potential of
Strychnos potatorum. Int. Res. J. Pharm. 2019; 10(9):204-210
http://dx.doi.org/10.7897/2230-8407.1009283
Source of support: Nil, Conflict of interest: None Declared
